Expression of cloned DNA in bacteria has been extensively studied. Most available information concerns expression of cloned native, foreign, and hybrid genes in E. coli using portable promoters in plasmids, phage or cosmid cloning vectors. Recently several laboratories have reported expression levels of 30% of total cellular protein as a single protein product.
The E. coli gene uidA encodes the enzyme .beta.-D-glucuronidase (E.C. 3.2.1.31) which is the first enzyme of the hexuronide-hexuronate pathway (Ashwell, G. [1962] Methods in Enzymol. 5:190-208). The enzyme is induced by .beta.-D-glucuronides but hydrolyzes both .beta.-D-glucuronides and the non-inducing .beta.-D-galacturonides to their respective uronic acids. Subsequent enzymes of the pathway convert glucuronate or galacturonate to 2-keto-3-deoxy-gluconate (KDG), which is in turn fed into the glycolytic pathway after phosphorylation to produce KDG-6-P and conversion to pyruvate plus glyceraldehyde-3-phosphate.
The uidA gene has been shown to be under negative regulation by the closely linked upstream repressor gene uidR and also under partial or weaker negative control by the uxuR gene (Ritzenthaler, P., Blanco, C. and Mata-Gilsinger, M. [1983] Mol. Gen. Genet. 191:263-270). uxuR is the repressor of the uxu operon which produces the enzymes necessary for the conversion of fructuronate to KGD later in the same pathway (Novel, M. and Novel, G. [1976] J. Bacteriol. 127: 407-417; ibid, 418-432).
Blanco et al. (1982, J. Bact. 149: 587-594) identified a plasmid U1, from the E. coli-ColE1 hybrid clone bank of Clarke and Carbon (1976, Cell 9: 91-99), which carries the manA, uidA, uidR region of the E. coli chromosome. When fully induced, strain JA200(U1) produced only 1.8-fold the .beta.-D-glucuronidase enzyme induced in JA200. In contrast, subclones which carried the uidA gene without a uidR repressor gene copy produced sufficient enzyme to be visualized by Coomassie blue staining of a non-denaturing polyacrylamide gel (FIG. 3 in Blanco et al.).